The present invention relates to a biocontrol agent against larvae of mosquitoes and blackflies, carrying a synergistic combination of endotoxin genes of Bacillus thuringiensis subsp. Israelensis. 
Bacillus thuringiensis subsp. israelensis has widely been used to control vectors of human infectious diseases such as mosquitoes and blackflies as described by World Health Organization. 1989 (Geographical distribution of arthropod-borne disease and their principle vectors. p. 249. In Vector Biology and Control Division, Geneva). Its larvicidal activity is contained in a parasporal crystalline inclusion synthesized during sporulation. A 75 MDa plasmid includes all the genes encoding xcex4-endotoxin proteins and their control elements as described by Hofte, H., (et. al. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53:242-255, 1989). As a biological pesticide, B. thuringiensis subsp. israelensis is widely accepted because of its specificity of action towards dipteran insects and lack of effects on other organisms. In addition, it is very unlikely that mosquitoes will develop resistance to the toxin because it is composed of several proteins.
Current application of B. thuringiensis subsp. israelensis for mosquito control is limited by its short half life under field conditions. One way to overcome this limitation is by cloning the genes involved in organisms which inhabit the mosquito breeding zones. Cyanobacteria has been considered as attractive candidates for this purpose as suggested by the present inventor and others, (Genetically engineered cyanobacteria as a BTI toxin genes delivery system: a biotechnological approach to the control of malaria mosquitoes, p. 49-64, in Proceeding of Combating Malaria UNESCO Conference 1995, Paris). Several attempts have been made during the last decade to produce genetically engineered mosquitocidal cyanobacteria. Most of the work seems in favor of cloning a single cry gene in unicellular cyanobacteria, but intact transformed cells displayed mosquito larvicidal activity below the level required for effective and practical use as bioinsecticides in a natural aquatic environment as reported (Soltes-Rak, E., et. al. Effect of promoter modification on mosquitocidal CryIVB gene expression in Synechococcus sp. strain PCC 7942. Appl. Environ. Microbiol. 59:2,404-2,410, 1993). Attempts to enhance expression of cryIV genes in cyanobacteria by strong promoters (e.g., PpsbA, PpsbB) were not satisfactory, probably because the transcription or translation product is unstable there (Soltes-Rak, E., et. al. ibid.).
The presence of P20 has been claimed to raise the levels of CytA, CryIVA and CryIVD in E. coli and in an acrystalliferous strain of B. thuringiensis, probably acting as a chaperone to stabilize them. Synergism among the three purified polypeptides (CryIVA, CryIVB, and CryIVD) was clearly demonstrated as described by S. poncet, et. al. (Evaluation of synergistic interactions among the CryIVA, CryIVB, and CryIVD toxic components of B. thuringiensis subsp. israelensis crystals. J. Invertebr. Pathol. 66:131-135, 1995), with the highest rate being reported between CryIVA and CryIVD.
With the above state of the art in mind, a combination of CryIVA, CryIVD and p20, previously cloned and expressed in E. coli as an operon (in pHE4-ADR) as described by the present inventor and others (Mosquito larvicidal activity of Escherichia coli with combinations of genes from Bacillus thuringiensis sbsp. israelensis. J. Bacteriol. 177: 2,851-2,857., 1995), was introduced into the nitrogen fixing filamentous Anabaena PCC 7120, and the most toxic clones selected for further studies.
In contradistinction to the results previously reported regarding attempts to enhance expression of CryIV genes in cyanobacteria it was found that there was produced a biocontrol agent having superior properties.
Thus, according to the present invention there is now provided a biocontrol agent against larvae of mosquitoes and blackflies comprising transgenic Anabaena PCC 7120 carrying a synergistic combination of the endotoxin genes CryIVA and CryIVD of Bacillus thuringiensis subsp. israelensis. 
While the invention will now be described in connection with certain preferred embodiments in the following examples and with reference to the appended figures so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention. dr
In the drawings:
FIG. 1 illustrates the construction of a shuttle vector for expression;
FIG. 2 graphically represents growth of exconjugant cells of Anabaena PCC 7120; and,
FIG. 3 graphically illustrates toxicities of Cry1VADR expressed in cyanobacterium Anabaena PCC 7120 carrying pSBJ2.